The overall goal of the proposed work is to understand the mechanism of assembly of the SOS ribosomal subunit. We have made a systematic set of deletions to 16S rRNA to generate fragments of the SOS subunit that correspond to domains, subdomains and assembly intermediates. Detailed thermodynamic , kinetic, and structural studies on these complexes will provide insights into the cooperative nature of protein binding and RNA conformational changes during SOS assembly. [unreadable] [unreadable] In the previous grant periods, we focused on the central domain of the SOS subunit, and we now extend our studies to include the 5'- and S'-domains. In addition, we have developed a stable isotope (15N) pulse-chase assay that allows us to monitor binding of all 20 proteins to the 16S rRNA simultaneously, using a mass spectrometric analysis. The Specific Aims for the current period are: [unreadable] [unreadable] 1) Understanding the assembly mechanism of the SOS central domain. [unreadable] [unreadable] 2) Biochemical analysis of the 5'- and S'-domains of the SOS subunit. [unreadable] [unreadable] 3) Monitoring assembly of the SOS subunit using isotope pulse-chase mass spectrometry. [unreadable] [unreadable] This supplemental application requests funding for an ultracentrifuge system and an LC-nanospray mass spectrometer that are essential to carry out the proposed studies. The demonstrated success of the isotope pulse chase assay now justifies the need for this additional equipment. [unreadable] [unreadable]